The activating role of phospho-(Tyr)-calmodulin on the epidermal growth factor receptor

The activity of calmodulin (CaM) is modulated not only by oscillations in the cytosolic concentration of free Ca2+, but also by its phosphorylation status. In the present study, the role of tyrosine-phosphorylated CaM [P-(Tyr)-CaM] on the regulation of the epidermal growth factor receptor (EGFR) has been examined using in vitro assay systems. We show that phosphorylation of CaM by rat liver solubilized EGFR leads to a dramatic increase in the subsequent phosphorylation of poly-L-(Glu:Tyr) (PGT) by the receptor in the presence of ligand, both in the absence and in the presence of Ca2+. This occurred in contrast with assays where P-(Tyr)-CaM accumulation was prevented by the presence of Ca2+, absence of a basic cofactor required for CaM phosphorylation and/or absence of CaM itself. Moreover, an antibody against CaM, which inhibits its phosphorylation, prevented the extra ligand-dependent EGFR activation. Addition of purified P-(Tyr)-CaM, phosphorylated by recombinant c-Src (cellular sarcoma kinase) and free of non-phosphorylated CaM, obtained by affinity-chromatography using an immobilized anti-phospho-(Tyr)-antibody, also increased the ligand-dependent tyrosine kinase activity of the isolated EGFR toward PGT. Also a CaM(Y99D/Y138D) mutant mimicked the effect of P-(Tyr)-CaM on ligand-dependent EGFR activation. Finally, we demonstrate that P-(Tyr)-CaM binds to the same site (645R-R-R-H-I-V-R-K-R-T-L-R-R-L-L-Q660) as non-phosphorylated CaM, located at the cytosolic juxtamembrane region of the EGFR. These results show that P-(Tyr)-CaM is an activator of the EGFR and suggest that it could contribute to the CaM-mediated ligand-dependent activation of the receptor that we previously reported in living cells.

An ethnopharmacological and historical analysis of “Dictamnus”, a European traditional herbal medicine

Ethnopharmacological relevance and background: “Dictamnus” was a popular name for a group of medicinal herbaceous plant species of the Rutaceae and Lamiaceae, which since the 4th century have been used for gynaecological problems and other illnesses BCE and still appear in numerous ethnobotanical records. Aims: This research has as four overarching aims: Determining the historical evolution of medical preparations labelled “Dictamnus” and the different factors affecting this long-standing herbal tradition. Deciphering and differentiating those medicinal uses of “Dictamnus” which strictly correspond to Dictamnus (Rutaceae), from those of Origanum dictamnus and other Lamiaceae species. Quantitatively assessing the dependence from herbal books, and pharmaceutical tradition, of modern Dictamnus ethnobotanical records. Determining whether differences between Western and Eastern Europe exist with regards to the Dictamnus albus uses in ethnopharmacology and ethnomedicine. Methods: An exhaustive review of herbals, classical pharmacopoeias, ethnobotanical and ethnopharmacological literature was conducted. Systematic analysis of uses reported which were standardized according to International Classification of Diseases – 10 and multivariate analysis using factorial, hierarchical and neighbour joining methods was undertaken. Results and discussion: The popular concept “Dictamnus” includes Origanum dictamnus L., Ballota pseudodictamnus (L.) Benth. and B. acetabulosa (L.) Benth. (Lamiaceae), as well as Dictamnus albus L. and D. hispanicus Webb ex Willk. (Rutaceae), with 86 different types of uses. Between 1000 and 1700 CE numerous complex preparations with “Dictamnus” were used in the treatment of 35 different pathologies. On biogeographical grounds the widespread D. albus is a far more likely prototypical “Dictamnus” than the Cretan endemic Origanum dictamnus. However both form integral parts of the “Dictamnus” complex. Evidence exists for a sufficiently long and coherent tradition for D. albus and D. hispanicus, use to treat 47 different categories of diseases. Conclusions: This approach is a model for understanding the cultural history of plants and their role as resources for health care. “Dictamnus” shows how transmission of traditional knowledge about materia medica, over 26 centuries, represents remarkable levels of development and innovation. All this lead us to call attention to D. albus and D. hispanicus which are highly promising as potential herbal drug leads. The next steps of research should be to systematically analyse phytochemical, pharmacological and clinical evidence and to develop safety, pharmacology and toxicology profiles of the traditional preparations.